A nerve injury–specific long noncoding RNA promotes neuropathic pain by increasing Ccl2 expression

Maladaptive changes of nerve injury–associated genes in dorsal root ganglia (DRGs) are critical for neuropathic pain genesis. Emerging evidence supports the role of long noncoding RNAs (lncRNAs) in regulating gene transcription. Here we identified a conserved lncRNA, named nerve injury–specific lncRNA (NIS-lncRNA) for its upregulation in injured DRGs exclusively in response to nerve injury. This upregulation was triggered by nerve injury–induced increase in DRG ELF1, a transcription factor that bound to the NIS-lncRNA promoter. Blocking this upregulation attenuated nerve injury–induced CCL2 increase in injured DRGs and nociceptive hypersensitivity during the development and maintenance periods of neuropathic pain. Mimicking NIS-lncRNA upregulation elevated CCL2 expression, increased CCL2-mediated excitability in DRG neurons, and produced neuropathic pain symptoms. Mechanistically, NIS-lncRNA recruited more binding of the RNA-interacting protein FUS to the Ccl2 promoter and augmented Ccl2 transcription in injured DRGs. Thus, NIS-lncRNA participates in neuropathic pain likely by promoting FUS-triggered DRG Ccl2 expression and may be a potential target in neuropathic pain management.

aortic cross clamp and transported to an onsite laboratory in an NMDG-based solution for tissue processing and primary cell dissociation and culture following protocols described in detail previously (43).

Animal models
For trauma-induced neuropathic pain models, sciatic nerve chronic constriction injury (CCI) and fourth lumbar (L4) spinal nerve ligation (SNL) in mice were carried out as described

Behavioral analysis
The evoked behavioral testing, including mechanical, heat, and cold tests, was carried out in sequential order at 1-hour intervals. Conditional place preference (CPP) testing was performed 2 or 4 weeks after surgery. Locomotor function testing was carried out before the tissue collection.
Mice were placed in a Plexiglas chamber on the cold aluminum plate, the temperature of which was monitored continuously by a thermometer. The paw withdrawal latency was recorded as the length of time between placement and the first sign of the mouse jumping and/or flinching. Each trial was repeated three times at 10-min intervals on the ipsilateral side. To avoid tissue damage, a cut-off time of 20 sec was used.
CPP test was carried out as described with minor modifications (23, 24, 44, 52). Briefly, an apparatus with two Plexiglas chambers connected with an internal door (Med Associates Inc., St. Albans, VT) was used. One of the chambers was made of a rough floor and walls with black and white horizontal stripes, and another one was composed of a smooth floor and walls with black and white vertical stripes. Movement of the mice and time spent in each chamber were monitored by photobeam detectors installed along the chamber walls and automatically recorded in MED-PC IV CPP software. Mice were first preconditioned for 30 min with full access to two chambers to habituate them to the environment. At the end of the preconditioning phase, basal duration spent in each chamber was recorded within 15 min to check whether animals had a preexisting chamber bias. Mice spending more than 80% or less than 20% of total time in any chamber were excluded from further testing. The conditioning protocol was performed for the following 3 days with the internal door closed. The mice first received an intrathecal injection of saline (5 µl) specifically paired with one conditioning chamber in the morning. Six hours later, lidocaine (0.8 % in 5 µl of saline) was given intrathecally paired with the opposite conditioning chamber in the afternoon. Lidocaine at this dosage did not affect motor function. The injection order of saline and lidocaine was switched every day. On the test day, at least 20 hours after the conditioning, the mice were placed in one chamber with free access to both chambers. The duration of time that each mouse spent in each chamber was recorded for 15 min. Score differences were calculated as test time-preconditioning time spent in the lidocaine chamber.
Locomotor function was examined as described (23,24,44,52). Three reflexes were conducted as follows. For the placing reflex, the placed positions of the hind limbs were slightly lower than those of the forelimbs, and the dorsal surfaces of the hind paws were brought into contact with the edge of a table. Whether the hind paws were placed on the table surface reflexively was recorded. For the grasping reflex, after the mouse was placed on a wire grid, whether the hind paws grasped the wire on contact was recorded. For the righting reflex, when the mice were placed on its back on a flat surface, whether it immediately assumed the normal upright position was recorded. Each trial was repeated 5 times at 5-min interval and the scores for each reflex were recorded based on counts of each normal reflex.

DRG microinjection
DRG microinjection was conducted as described with minor modification (53-55). Briefly, after the mouse was anesthetized with isoflurane, a dorsal midline incision was made in the lower lumbar back region. The unilateral L4 and/or L3 articular processes were exposed and then removed. Viral solution (1 µl/DRG, 4-9 × 10 12 ) or siRNA solution (1 µl/DRG, 40-80 µM) was injected into ipsilateral exposed L4 or L3/4 DRGs with the use of a glass micropipette connected to a Hamilton syringe. After injection, the 10 min pipette retention was used before it was removed. The surgical field was irrigated with sterile saline and the skin incision closed with wound clips. None of the microinjected mice showed signs of paresis or other abnormalities. Injected DRGs that were stained with hematoxylin/eosin confirmed the integrity of their structure and demonstrated no visible leukocytes. were euthanized with isoflurane, all DRGs were collected in cold Neurobasal Medium (Gibco/ThermoFisher Scientific) containing 10% fetal bovine serum (JR Scientific, Woodland, CA), 5 mL L-glutamine (200 mM) (Gibco/ThermoFisher Scientific), 10 mL B-27® Supplement (50x) (Gibco/ThermoFisher Scientific), 100 units/ml Penicillin and 100 µg/ml Streptomycin (Quality Biological, Gaithersburg, MD). The DRGs were then treated with enzyme solution (5 mg/ml dispase, 1 mg/ml collagenase type I) in Hanks' balanced salt solution (HBSS) without Ca 2+ and Mg 2+ (Gibco/ThermoFisher Scientific). After trituration and centrifugation, dissociated cells were resuspended in mixed Neurobasal Medium and plated in a six-well plate coated with 50 µg/ml poly-D-lysine (Sigma, St. Louis, MO) at 1.5-4 × 10 5 cells. The cells were incubated at 5% CO2 and 37 ℃. On the second day, 4-10 µl of virus (titer ≥ 6-9 × 10 12 /µl), 300-500 ng of vector or siRNA (100 nM; transfected with Lipofectamine 2000) was added to each 2 ml well. Cells were collected 3 days later.

Quantitative real-time reverse transcription (RT)-PCR
The unilateral L3/4 DRGs from two adult CCI, corresponding sham surgery, incisional, CFA, or MIA mice, the unilateral L4 DRGs from four adult SNL or corresponding sham mice, or the cultured DRGs neurons from one well of a 6-well plate were collected rapidly and pooled together to achieve enough RNA. Total RNA was extracted by the RNeasy Mini Kit (Qiagen, Valencia, CA) and treated with excess DNase I (New England Biolabs, Ipswich, MA). Highly purified, DNase-treated RNA samples from human DRG were purchased from Clontech Laboratories, Inc. (Mountain View, CA). RNA concentration was measured using the NanoDrop 2000 Spectrophotometer (Thermo Scientific, Wilmington, DE). Ratios of A260/280nm were between 1.97 and 2.08. RNA (500 ng) was reverse-transcribed into single-stranded cDNA using the Omniscript RT Kit (Qiagen) with specific RT-primers or oligo(dT) primers, and the cDNA template was amplified by real-time PCR using the primers listed in the Supplemental Table 3.
Each sample was run in triplicate in a 20 μL reaction using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA). Reactions were carried out in a BIO-RAD CFX96 real-time PCR system. Ratios of RNA levels at different time points post-surgery (or post-injection) to RNA levels at 0 hours (or days) before surgery/injection, or of RNA levels in other treated groups to RNA levels in the control group were calculated using the ΔCt method (2 −ΔΔCt ). All data were normalized to Tuba1α, which was stable even after peripheral nerve injury insult in mice as shown in our previous studies(23, 24, 44, 52).

Single-cell RT-PCR
The freshly dissociated DRG neurons from adult mice or donors were first prepared as described (23, 24, 43, 44, 52). Briefly, 4 hours (for mouse DRG culture ) or 12-20 hours (for human DRG culture) after plating, single mouse living large (> 35 m), medium (25-35 m), and small (< 25 m) DRG neurons (56) or single human living large (> 60 µm) and small (≤ 60 µm) DRG neurons (57) were sorted under an inverted microscope fit with a micromanipulator and microinjector and collected in a PCR tube with 6-8 μl of cell lysis buffer (Signosis, Sunnyvale, CA). After centrifugation, the supernatants were collected and divided into PCR tubes for different genes. The remaining RT-PCR procedure was performed according to the manufacturer's instructions with the Single-Cell RT-PCR Assay Kit (Signosis). All nest-PCR primers used are listed in Supplemental Table 3.

Rapid amplification of cDNA ends (RACE)
To determine the 5' and 3' end of both NIS V1 and NIS V2, the 5' and 3' RACE were carried out using a 2nd Generation 5'/3' RACE Kit (Roche Diagnostics, Indianapolis, IN). For the 5' RACE, the cDNA of each variant was first reversely transcribed from the total RNA of mouse DRG by strand-specific primers followed by poly(A) tailing and PCR amplification of the 5'-end of cDNA according to the manufacturer's instructions. The 3' RACE analysis was performed by reverse transcription of cDNA using oligo dT-anchor primer followed by genespecific and anchor primer amplification. All primers are listed in Supplemental Table 3. PCR products from 5' RACE and 3' RACE were extracted, purified, and cloned to pCRBlunt II-TOPO vector (ThermoFisher) for DNA sequencing. All sequences were analyzed and the fulllength NIS V1 and NIS V2 were determined.

RNA fractionation
Separation of nuclear and cytoplasmic fractions of primary cultured mouse DRG neurons followed by RNA isolation was carried out by using PARIS Kit (Invitrogen) (58). Briefly, after being rinsed with 1× DPBS, the cultured DRG neurons were lysed in ice-cold cell fractionation buffer. After incubation on ice for 10 minutes, the lysate was centrifuged for 1,000 × g at 4°C for 10 minutes to separate the nuclear and cytoplasmic fractions. Total RNA of each fraction was extracted by following the Kit instructions. Various gene/transcript expression levels in both nuclear and cytoplasmic fractions of all samples were quantified by quantitative real-time RT-PCR as described above.

Plasmid constructs and virus production
Full-length Elf1, Fus, NIS V1 and NIS V2 cDNAs were respectively amplified from mouse DRG RNA by using the SuperScript III One-Step RT-qPCR System with the Platinum Taq High Fidelity Kit (Invitrogen/Thermo-Fisher Scientific) and primers with restriction enzymes (Supplemental Table 3). After double enzyme digestion, the PCR products were inserted into the corresponding sites of the pHpa-tra-SK plasmids (University of North Carolina, Chapel Hill) to replace enhanced GFP sequence or the multiple cloning site of the pAAV-MCS vector (Cell Biolabs, CA). The resulting vectors expressed the genes under the control of the cytomegalovirus promoter. The designed sense and antisense sequences for Elf1 shRNA and Fus shRNA were annealed and inserted between the BamHI and XbaI sites of pAAV-shRNA-EF1a-EYFP. AAV5 packaging of viral particles carrying the cDNA was carried out using the AAVpro Purification Kit (Takara, Mountain View, CA). Virus titer was evaluated using the AAVpro® Titration Kit (Takara). AAV5-Cre and AAV5-Gfp were purchased from UNC Vector Core.

Northern blotting
To prepare complementary RNA (cRNA) probes of mouse NIS V1 and NIS V2, two PCR products with 400 bp and 452 bp fragments were amplified using mouse cDNA with a pair of primers including the T7 promoter at the 3' end (Supplemental Table 3) and identified using DNA sequencing. After PCR purification, a riboprobe was generated through in vitro transcription and labeled with digoxigenin-dUTP according to the manufacturer's instructions (Roche Diagnostics, Indianapolis, IN) at 37 °C for 2 h. The probes were purified using Micro Bio-Spin TM 30 Chromatography Column (Bio-Rad).
Northern blot analysis was performed as described previously (24, 59). Briefly, the RNA (10 μg) extracted from injured DRGs 7 days after SNL was separated on an agarose/formaldehyde gel (2% for NIS V1 and 1% for NIS V2), transferred to a BrightStar-plus positively charged nylon membrane, and cross-linked using UV light. After pre-hybridization, the membrane was hybridized overnight at 68°C with a digoxigenin-UTP-labeled cRNA probe for NIS-lncRNA. The membrane was washed in a low salt buffer at room temperature for 2 × 5 min, high salt buffer at 68 °C for 2 × 5 min and SSC at 68 °C for 1 × 2 min. After being blocked, the membrane was incubated with alkaline phosphatase-conjugated sheep anti-digoxigenin (1:500, Roche) for 1 h at room temperature, and washed for 2 × 5 min, incubated by CDP-Star solution provided in the DIG Northern Starter Kit (Roche) and imaged by the ChemiDoc XRS System with Image Lab software (Bio-Rad). To clearly display the markers, the membranes loaded with the markers were separately imaged.

Luciferase reporter assay
A 693-bp fragment from the NIS-lncRNA promoter region (including the ELF1-binding motif) and a 1,664-bp fragment from the Ccl2 promoter region (including FUS-binding sites) were amplified by PCR from genomic DNA using primers (Supplemental Table 3) to construct the NIS-lncRNA and Ccl2 gene reporter plasmids, respectively, as described previously (24). The PCR products were cloned into the KpnI/HindIII or KpnI/NheI restriction sites of pGL3-Basic vector (Promega, Madison, WI). The accuracy of recombinant clones was verified by DNA sequencing. HEK-293T or CAD cells were plated on a 12-well plate and cultured at 37 °C in a humidified incubator with 5% CO2. One day after culture, the cells in each well were cotransfected with 300 ng of plasmid (expressing full-length Elf1, Fus, NIS V1 or NIS V2), 300 ng of pGL3-Basic vector with or without the reporter plasmid, and 10 ng of the pRL-TK (Promega) using Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. The wells were divided into different groups as indicated. Two days after transfection, the cells were collected and lysed in passive lysis buffer. Approximately 10 μl of supernatant was used to measure the luciferase activity using the Dual-Luciferase Reporter Assay System (Promega).
Transfection experiments were repeated 3 independent times. Relative reporter activity was calculated after normalization of the firefly activity to renilla.

RNA sequencing
The ipsilateral L3/4 DRGs were harvested 5 weeks after microinjection with AAV5-Gfp or a mixture of AAV5-V1 and AAV5-V2 into the unilateral L3/4 DRGs of mice. Eight DRGs from four microinjected mice were pooled together to achieve enough RNA. Total RNA (1.2 µg/sample) extracted as described above was subjected to rRNA depletion by Ribo-Zero rRNA Immunoreactive positive neurons containing three or more particles of NIS V1 or NIS V2 were considered to be "co-expressed" cells, according to preceding studies(60, 61) and counted manually.

Bioinformatics prediction of transcription factors
The University of Santa Cruz (UCSC) genomic database (https://genome.ucsc.edu) was used to acquire the 2,000-bp promoter sequence of NIS-lncRNA gene. JASPAR database (http://jaspar.genereg.net/) was used to predictively analyze whether there were motifs of ELF1 in the promoter region of the NIS-lncRNA gene. The relative profile score threshold was set as 80%. Through analyzing the predicted score from the potential ELF1-binding regions, the region with the highest score was used to clone and to investigate the binding between ELF1 and NIS-lncRNA.

In vitro protein translation
The full-length NIS V1 and NIS V2 DNA fragments containing the T7 promoter were obtained by RT-PCR with the primers listed in Supplemental Table 3 and were in vitro transcribed and translated by the Transcend™ Non-Radioactive Translation Detection System procedures described in the manufacturer's manual (Promega, Madison, WI). In this system, biotinylated lysine residues were incorporated into nascent proteins during translation, allowing for in vitro non-radioactively-labeled protein synthesis. Proteins were detected by incubation with streptavidin-horseradish peroxidase and visualized using Western blotting procedure (62).
Luciferase and Creb1 were used as coding gene controls.

RNA pull down and mass spectrometry
The RNA-protein pull down assay was performed as described previously (44, 63). The cultured DRG neurons from one-month mice were prepared as mentioned above. Three days after culture, DRG neurons were rinsed with chilled PBS and collected in Pierce IP Lysis Buffer Proteomics, only those proteins pulled down by sense probes, but not negative control antisense probes, were considered to be the positive targeted proteins. To verify the binding of NIS-lncRNA to FUS, Western blot analysis was carried out as described below.

Chromatin isolation by RNA purification
Chromatin isolation by RNA purification was carried out as reported (63,64). Briefly, the DRG neuronal culture was prepared as described above. Three days after culture, the neurons were rinsed with chilled PBS and crosslinked by 1% formaldehyde for 10 min. The reactions were quenched by adding 0.125 M glycine for 5 min. After cell pellets were dissolved in nuclear lysis buffer, the cell lysates were sonicated to break DNA to 100-500 bp fragments. A total of 14 different biotinylated antisense DNA probes that were complementary to the sequence of NIS-lncRNA were designed using an online tool (Singlemoleculefish.com) and numbered. The negative control probes that were not complementary to the sequence of NIS-lncRNA were used as a negative control. Seven odd-numbered probes, seven even-numbered probes and negative control probes were hybridized, respectively, with the cell lysates overnight. The complex of beads/probes/DNA was pulled down by using streptavidin magnetic C1 beads (Invitrogen). The DNA was collected with pre-spin down yellow phase-lock gel tubes and the potential binding DNA was ready for analysis by quantitative PCR assay as described above.

RNA immunoprecipitation (RIP) Assay
The RIP assay was conducted using the Magna RIP Kit (Upstate/ EMD Millipore, Darmstadt, Germany) as described previously (44). The homogenates from mouse SNL or sham DRGs were suspended in the RIP lysis buffer containing the protease inhibitor cocktail and RNase inhibitor.
The RIP lysate was incubated on ice for 5 min and stored at -80℃. The Magnetic Beads Protein A/G suspension for each IP was washed twice with the RIP wash buffer. Mouse anti-FUS antibody (2.0 µg; catalog number: AB154141, Abcam) or purified mouse IgG was conjugated to Magnetic Beads Protein A/G re-suspended in RIP wash buffer for 30 min at room temperature.
After being washed three times with RIP wash buffer, the Beads Protein A/G-antibody complexes were re-suspended into the RIP immunoprecipitation buffer. After being thawed and centrifuged at 14,000 rpm at 4℃ for 10 min, the supernatants of the RIP lysate were incubated with beads-antibody complex in the RIP immunoprecipitation buffer overnight at 4℃ by rotating. After the samples were washed six times with the RIP wash buffer, RNA was eluted from the beads by incubating in the proteinase K buffer at 55℃ for 30 min by shaking, purified by phenol/chloroform extraction and analyzed by quantitative RT-PCR as described above. The supernatant of the RIP lysate was used as Input. All primers used are listed in Supplemental Table 3.

Chromatin immunoprecipitation (ChIP) assay
Chromatin immunoprecipitation was performed using the EZ ChIP Kit (Upstate/EMD Millipore, Darmstadt, Germany) as described previously (23, 24, 65). Briefly, DRG homogenates were crosslinked with 1% formaldehyde for 10 min at room temperature. The reaction was terminated by the addition of 0.25 M glycine. After centrifugation, the collected pellet was lysed by SDS lysis buffer with a protease inhibitor cocktail and sonicated until the DNA was broken into fragments with a mean length of 200-1,000 bp. After the samples were pre-cleaned with protein G agarose, they were subjected to immunoprecipitation overnight with 2 μg of rabbit anti-ELF1 (catalog number: ORB315774, Abcam), mouse anti-FUS (catalog number: AB154141, Abcam), or purified rabbit IgG overnight at 4 °C. Input (10-20% of the sample for immunoprecipitation) was used as a positive control. The DNA fragments were purified and identified using PCR/quantitative real-time PCR with the primers listed in Supplemental Table 3.

Western blotting
To achieve sufficient protein, unilateral L3/4 DRGs from 2 CCI/sham mice, or unilateral L4 DRGs from 4 SNL/sham mice were pooled together. The tissues were homogenized and the cultured cells ultrasonicated in chilled lysis buffer (10 mM Tris, 1 mM phenylmethylsulfonyl fluoride, 5 mM MgCl2, 5 mM EGTA, 1 mM EDTA, 1 mM DTT, 40 μ M leupeptin, 250 mM sucrose). After centrifugation at 4 °C for 15 min at 1,000 g, the supernatant was collected for cytosolic/membrane proteins and the pellet for nuclear proteins. The protein concentration in the samples was measured using the Bio-Rad protein assay (Bio-Rad) and the samples then heated at 99 °C for 5 min and loaded onto a 4-15% stacking/7.5% separating SDS-polyacrylamide gel (Bio-Rad Laboratories). The proteins were then electrophoretically transferred onto a polyvinylidene difluoride membrane (Bio-Rad Laboratories). After the membranes were blocked with 3% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 for 1 h, they were incubated overnight at 4 °C with the following primary antibodies including goat anti-ELF1 Signaling), and rabbit anti-histone H3 (1:1,000, catalog number: 17168-1-AP, Proteintech). The proteins were detected by horseradish peroxidase-conjugated anti-mouse secondary antibody (1:3,000, catalog number: 17168-1-AP, Jackson ImmunoResearch) or anti-rabbit secondary antibody (1:3,000, catalog number: 115-035-003, Jackson ImmunoResearch) and visualized by western peroxide reagent and luminol/enhancer reagent (Clarity Western ECL Substrate, Bio-Rad) and exposed using the ChemiDoc XRS System with Image Lab software (Bio-Rad). The intensity of blots was quantified with densitometry using Image Lab software (Bio-Rad). All cytosolic/membrane protein bands were normalized to GAPDH and all nuclear proteins to histone H3.

Whole-cell patch-clamp recording
The DRG neurons were and recorded, respectively. The resting membrane potential was taken 3 min after a stable recording was first obtained. Action potential (AP) was evoked by delivering depolarizing currents. The membrane potential was held at the existing resting membrane potential during the current injection. The rheobase current was defined as the first step current which induced one AP by 50 ms depolarizing step current. Small and medium DRG neurons were injected between 0 pA and 120 pA in 10 pA increments and large DRG neurons were injected between 100 pA and 600 pA in 50 pA increments, in current-clamp mode. The spike frequency versus injected current experiments were performed by measuring the average action potential firing rate during 500 ms depolarizing step current injection. Small and medium DRG neurons ranged between 20 pA and 160 pA in 20 pA increments and large DRG neurons ranged between 100 pA and 800 pA in 100 pA increments in current-clamp mode. The AP amplitude was measured between the peak and the baseline and the AP overshoot was measured between the AP peak and 0 mV. The after-hyperpolarization amplitude was measured between the maximum hyperpolarization and the final plateau voltage. The number of spontaneous APs was calculated as the number of APs per second in 3 min during extracellular buffer application.

Statistical analysis.
For in vivo experiments, mice were distributed into various treatment groups randomly. For in vitro experiments, the cells were evenly suspended and then randomly distributed into each well tested. The sample sizes were determined based on our pilot studies, previous reports in the field (24, 45, 70-72) and power analyses (power of 0.90 at p < 0.05). All of the results are given as means ± S.E.M. Data distribution was assumed to be normal but this was not formally tested.
The data were statistically analyzed using two-tailed, paired Student's t-test and a one-way, twoway, or three-way ANOVA. When ANOVA showed a significant difference, pairwise comparison between means was performed using the post hoc Tukey method (SigmaPlot 12.5, San Jose, CA). Significance was set at P < 0.05.